The NAC Family Transcription Factor Gmnac42–1 Regulates
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Jahan et al. BMC Genomics (2019) 20:149 https://doi.org/10.1186/s12864-019-5524-5 RESEARCHARTICLE Open Access The NAC family transcription factor GmNAC42–1 regulates biosynthesis of the anticancer and neuroprotective glyceollins in soybean Md Asraful Jahan1, Brianna Harris2, Matthew Lowery3, Katie Coburn2, Aniello M. Infante4, Ryan J. Percifield2, Amanda G. Ammer5 and Nik Kovinich1* Abstract Background: Glyceollins are isoflavonoid-derived pathogen-inducible defense metabolites (phytoalexins) from soybean (Glycine max L. Merr) that have important roles in providing defense against pathogens. They also have impressive anticancer and neuroprotective activities in mammals. Despite their potential usefulness as therapeutics, glyceollins are not economical to synthesize and are biosynthesized only transiently and in low amounts in response to specific stresses. Engineering the regulation of glyceollin biosynthesis may be a promising approach to enhance their bioproduction, yet the transcription factors (TFs) that regulate their biosynthesis have remained elusive. To address this, we first aimed to identify novel abiotic stresses that enhance or suppress the elicitation of glyceollins and then used a comparative transcriptomics approach to search for TF gene candidates that may positively regulate glyceollin biosynthesis. Results: Acidity stress (pH 3.0 medium) and dehydration exerted prolonged (week-long) inductive or suppressive effects on glyceollin biosynthesis, respectively. RNA-seq found that all known biosynthetic genes were oppositely regulated by acidity stress and dehydration, but known isoflavonoid TFs were not. Systemic acquired resistance (SAR) genes were highly enriched in the geneset. We chose to functionally characterize the NAC (NAM/ATAF1/2/CUC2)-family TF GmNAC42–1 that was annotated as an SAR gene and a homolog of the Arabidopsis thaliana (Arabidopsis) indole alkaloid phytoalexin regulator ANAC042. Overexpressing and silencing GmNAC42–1 in elicited soybean hairy roots dramatically enhanced and suppressed the amounts of glyceollin metabolites and biosynthesis gene mRNAs, respectively. Yet, overexpressing GmNAC42–1 in non-elicited hairy roots failed to stimulate the expressions of all biosynthesis genes. Thus, GmNAC42–1 was necessary but not sufficient to activate all biosynthesis genes on its own, suggesting an important role in the glyceollin gene regulatory network (GRN). The GmNAC42–1 protein directly bound the promoters of biosynthesis genes IFS2 and G4DT in the yeast one-hybrid (Y1H) system. Conclusions: Acidity stress is a novel elicitor and dehydration is a suppressor of glyceollin biosynthesis. The TF gene GmNAC42–1 is an essential positive regulator of glyceollin biosynthesis. Overexpressing GmNAC42–1 in hairy roots can be used to increase glyceollin yields > 10-fold upon elicitation. Thus, manipulating the expressions of glyceollin TFs is an effective strategy for enhancing the bioproduction of glyceollins in soybean. Keywords: Phytoalexin, Transcription factor, NAC, Isoflavonoids, Glyceollins * Correspondence: [email protected] 1Division of Plant and Soil Sciences, West Virginia University, Morgantown, West Virginia 26506, USA Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Jahan et al. BMC Genomics (2019) 20:149 Page 2 of 21 Background AgNO3 was largely independent of ethylene signaling In 1939 K.O. Mueller et al. reported that metabolites [21]. Herbicides such as acifluorfen elicit at least in that were elicited in potato upon inoculation with an in- part via the reactive oxygen species (ROS) signaling compatible race of Phytophthora infestans subsequently pathway(s). The ups1 loss-of-function mutant of Arabi- provided resistance to a compatible race [1]. Since then, dopsis defective in ROS signaling had reduced camalexin the pathogen-inducible defense metabolites that have levels in response acifluorfen [23]. ups1 also had reduced been identified from numerous plant species have col- camalexin levels in response to Pseudomonas syringae and lectively been referred to as ‘phytoalexins’. Some phyto- P. syringae pv maculicola (Psm), suggesting a shared biotic alexins have essential roles in defending agricultural and abiotic elicitation pathway. In soybean, treatments crops against major pathogens. A classic example is the with JA, ethylene, P. sojae WGE, or hydroxyl radical (a glyceollins of soybean that provide resistance to the ROS) were highly effective at priming glyceollin biosyn- oomycete Phytophthora sojae [2–4]. For decades re- thesis in cells distal to the point of treatment, whereas SA searchers have studied the genetic regulation of phyto- was not [23, 24]. alexin elicitation by pathogens. Efforts have recently In contrast to the abiotic stresses and signaling mole- focused on identifying the transcription factors (TFs) cules that have conserved roles in eliciting phytoalexins that activate phytoalexin biosynthesis, a goal that has in response to abiotic stresses, the TFs found to regulate been confounded by the myriad of plant responses that phytoalexin biosynthesis have varied widely among plant occur synchronously in response to pathogens. Phyto- species. GaWRKY1 activated gossypol biosynthesis in alexins are biosynthetically diverse among plant species cotton [8]. GaWRKY1 transcripts were induced by methyl and include the isoflavonoid-derived glyceollins from jasmonate (MeJA) and Verticillium dahlia but not by SA soybean, the phenylpropanoid stilbenes from grapevine, or H2O2. GaWRKY1 transcripts were co-expressed both the phenolic aldehyde gossypol from cotton, the terpenoid spatially and temporally with gossypol biosynthesis genes momilactones and phytocassanes from rice, and the indole and GaWRKY1 was able to directly bind the promoter of alkaloid camalexin from Arabidopsis [5–10]. Since the TFs (+)-δ-cadinene synthase (CAD1)intheY1Hsystem.An- that activate the biosynthesis of phytoalexins in different other WRKY-family TF, namely AtWRKY33, was identified plant species belong to different gene families and/or are from Arabidopsis to directly bind and activate the promoter non-homologous, for decades an important question has of the camalexin biosynthesis gene PAD3 [25]. WRKY33 remained whether phytoalexin TFs are as diverse as the transcripts were induced by the ROS-inducing herbicide biosynthetic pathways that they regulate. Yet, several ex- paraquat, SA, and necrotrophic fungal pathogens [10]. cellent reviews highlight that phytoalexins share common GaWRKY1 and AtWRKY33 were not homologous since abiotic elicitors [11–13]. This could suggest conserved the proteins they encode had more than 20 other proteins regulatory pathways and TFs among plant species despite that were more similar by reciprocal BLASTPs. the biosynthetic heterogeneity of phytoalexins. The R2R3-type MYB TF genes VvMYB14 and VvMYB15 Highly conserved abiotic elicitors of phytoalexins in- from grapevine were co-induced with stilbene biosynthesis clude heavy metals, herbicides, and UV irradiation. genes in response to UV irradiation, wounding, and the UV elicits stilbene phytoalexins in grapevine, Cissus pathogen Plasmopara viticola [26]. The proteins directly Antarctica,andCannabis sativa [14], the flavonoid bound the promoter of STILBENE SYNTHASE (STS)in and diterpenoid phytoalexins in rice [15, 16], camalexin in transient gene reporter assays using grapevine suspension Arabidopsis [17], and glyceollins in soybean [18]. In rice, cells and induced the accumulation of stilbenes when over- loss-of-function mutants of the JA biosynthesis gene allene expressed in grapevine hairy roots [26]. Homologs of oxide cyclase (aos) or jasmonic acid-amido synthetase VvMYB14 and VvMYB15 in Arabidopsis did not regulate (osjar1–2) resulted in an almost complete loss of sakurane- camalexin biosynthesis but rather cold tolerance and tin elicitation in response to UV [19]. Yet, the diterpenoid defense-induced lignification, respectively [27, 28]. Double phytoalexins of rice were not affected in JA biosynthesis and triple mutants of the Arabidopsis R2R3 MYBs mutants. Copper chloride (CuCl2) elicitation of sakuranetin, AtMYB34, AtMYB51,andAtMYB122 had reduced cama- momilactone, and diterpenoid phytoalexins in rice was lexin levels upon elicitation with UV, AgNO3, and a PAMP dramatically reduced by JA biosynthesis inhibitors [20]. The isolated from Pythium aphanidermatum (PaNie)[29]. heavy metal silver nitrate (AgNO3) elicited glyceollin However, these three MYBs were unable to bind camalexin accumulation in soybean by reducing its degradation and biosynthesis gene promoters and feeding the triple mutant by enhancing the hydrolysis of isoflavone-glycoside conju- plant with a biosynthetic intermediate restored camalexin gates that compete with glyceollins for the common biosyn- accumulation, suggesting that AtMYB34, AtMYB51,and thetic intermediate daidzein [21]. AgNO3 was shown to AtMYB122 did not regulate